Department of Chemistry, California State University Fresno, Fresno, California.
Protein Sci. 2020 Apr;29(4):1004-1017. doi: 10.1002/pro.3831. Epub 2020 Feb 8.
A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (T = 12°C lower than wild-type), and a loss of the ability to fold reversibly due to heat induced aggregation. X-ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B-factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.
骆驼（Camelus dromedarius）VHH 结构域的一个显著特征是非典型的 CDR1 和 CDR3 之间的二硫键。该二硫键可能提供了一种进化优势，因为其中一个键合的半胱氨酸是胚系编码的。有人假设，这种额外的二硫键可能通过减少与抗原结合时长 CDR3 环固定相关的熵罚来发挥作用，从而提高结合亲和力。为了研究非典型二硫键对抗原结合的作用和 VHH 结构域的生物物理特性，我们使用了 R303 VHH，它作为模型结合李斯特菌毒力因子 InlB。通过定点突变，我们产生了 R303 的双突变体（C33A/C102A），以去除 VHH R303 的额外二硫键。抗原结合不受二硫键缺失的影响，然而突变体 VHH 的热稳定性降低（比野生型低 12°C），并且由于热诱导聚集而失去可逆折叠的能力。单独和与 InlB 复合物的突变体的 X 射线结构显示结构没有明显变化。结构的 B 因子分析表明，二硫键的缺失没有引起 CDR 环灵活性的重大变化，并且在抗原结合时没有发现环固定的证据。这些结果表明，骆驼 VHH 中发现的非典型二硫键可能已经进化到稳定结构域的生物物理特性，而不是在抗原结合中发挥重要作用。
